Aim: To investigate the occurrence of ESBL and colistin-resistant E. coli and its genotypic characterization and identification of virulence determinants in cases of bovine mastitis. Materials & methods: Milk samples presenting mastitis symptoms were screened for the presence ESBL, colistin-resistant, and different virulence genes. The clonal relationship was then evaluated with MLST, MLSA, and PFGE. Results: Genotypic characterization showed a dominance of blaCTX-M-1 group, and the most prevalent alleles observed were blaCTX-M-28 (38.37%), blaCTX-M-14 (17.44%), blaCTX-M-66 (13.95%), and blaCTX-M-55 (10.46%). The targeted virulence genes were detected in 97.89% isolates. The majority of the E. coli isolates carrying ESBL, and mcr-1 were clonally unrelated. Conclusion: High level of association was observed between ESBL-producing and COL-resistance in E. coli of bovine mastitis.